Project Summary Staphylococcus aureus is a Gram-positive, opportunistic pathogen that causes severe nosocomial and community-acquired pneumonia. In the lung, S. aureus encounters epithelial cells and macrophages, and recent reports suggest these cells serve as a replication niche. However, conflicting information exists regarding the ability of S. aureus to survive and replicate in host cells, specifically when comparing host species and bacterial clonal lineages of differing degrees of virulence. Numerous studies have used animal models of infection and human cell lines to study bacterial proliferation and the pro-inflammatory response to S. aureus; however, these models do not entirely mimic the human condition due, in part, to critical virulence factors with species specificity. The current proposal will establish a novel infection system that accurately mimics the interaction between S. aureus and human lungs. Human lung tissue and isolated pulmonary cells will be exposed to clinically relevant S. aureus isolates to investigate the pathogen's cellular replication niche and the host response to infection. Aim 1 will characterize the target niche of S. aureus in the human lung and assess bacterial replication in distinct cell types. Aim 2 will define the role of two S. aureus cytotoxins in triggering a pro- inflammatory innate immune response and disrupting tissue integrity and cell survival. This Aim will use ?-toxin and Panton-Valentine Leukocidin as model virulence factors to establish our ex vivo infection system as a disease-relevant method for defining virulence factor activity. Collectively, the proposed studies will establish a human disease relevant infection platform to study pulmonary S. aureus infection and virulence factor activity.